In nature, Francisella tularensis causes the zoonotic disease tularemia. F. tularensis is a category A select agent because of its highly infectious nature and the potential ease of its dissemination by aerosolization. Worldwide terrorist activities have resulted in growing concerns about the potential use of F. tularensis as a bioterrorist weapon; in response, the National Institute of Allergy and Infectious Diseases has expanded its biodefense research program in an effort to develop new F. tularensis therapeutics and vaccines. The long term goal of the proposed research is to identify novel targets for the development of tularemia vaccines and therapeutics. However, current genetic research is limited due to a shortage of selectable genetic markers for use in virulent F. tularensis strains. In this proposal we will develop non-antibiotic markers based on amino acid prototrophy for use as positive selective genetic markers in F. tularensis. We will use a genetic approach to identify heterologous genes that complement F. tularensis strain Schu S4 for growth on defined media lacking essential amino acids. The resulting genes will be used to generate several new F. tularensis shuttle vectors. The results of the proposed work will provide much needed genetic tools which will facilitate drug discovery, vaccine development, cell biology experiments, and microbiological experiments with virulent F. tularensis strains. [unreadable] [unreadable] [unreadable]